This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 4755–4757 4755

Cite this: Chem. Commun., 2012, 48, 4755–4757

Photocaged permeability: a new strategy for controlled drug releasew

M. Michael Dcona,ab

Deboleena Mitra,ab

Rachel W. Goehe,bc

David A. Gewirtz,bc

Deborah A. Lebmanbd

and Matthew C. T. Hartman*ab

Received 5th February 2012, Accepted 7th March 2012

DOI: 10.1039/c2cc30819c

Light is used to release a drug from a cell impermeable small

molecule, uncloaking its cytotoxic effect on cancer cells.

Off-target toxicity plagues conventional cancer chemotherapy. One

strategy to enhance selectivity of anti-cancer drugs involves un-

masking the cytotoxicity of a molecule in the vicinity of the tumor.1

This type of activation can be mediated by enzymes,2,3 changes in

pH,4 or exogenous factors such as temperature5 or light.6–8

Light is an ideal external stimulus since it provides a broad

range of adjustable parameters9 that can be optimized for

biological compliance. Several approaches that use light for

biomolecular activation have been reported.10–15 One established

method to enable selectivity of drug action using light is photo-

dynamic therapy (PDT). In PDT,16,17 light activation of a

photosensitizer generates cytotoxic singlet oxygen killing only

illuminated cells. PDT is currently used in several types of

malignancies16 including skin, lung, esophageal, bladder, head

and neck, and prostate cancer. A related strategy, photochemical

internalization,18,19 also uses photosensitizers. In this case, the

photosensitizers are used to release macromolecular cytotoxins

from endosomes, enabling their entry into the cytosol. However,

these approaches suffer from disadvantages,20 including unpre-

dictable drug uptake rates, the limited diffusion and lifetime of1O2, and the requirement for moderate levels of O2 which may

not always be available in the tumor environment.

In this communication, we report a new light-targeted drug

delivery system, which operates independently of the creation

of 1O2. The basis of the system is the attachment of a cell

impermeable small molecule to a drug via a linker that can be

removed in presence of light, allowing cellular entry (Fig. 1).

We call this new strategy photocaged permeability (PCP).21

More specifically, we report the controlled release of the anti-

cancer drug, doxorubicin (Dox) (Fig. 2), upon illumination.

To prevent entry of Dox in the dark we attached Dox to

EDANS (Fig. 2), a small fluorophore, chosen because it contains

a sulfonic acid moiety known to hinder cellular entry.22 To

connectDox to EDANSwe utilized a light-cleavable nitroveratryl23

linker. The nitroveratryl moiety has been used previously as a

photocaging group for a wide variety of biomolecules.24

The synthesis (Scheme 1) began with commercially available

nitroveratryl carboxylic acid (1). The N-hydroxy succinimide

ester was prepared followed by coupling with propargylamine

to give amide (2). This compound was converted to the

p-nitrophenyl carbonate which was then treated with doxo-

rubicin3 to generate photocaged doxorubicin carbamate (3).

EDANS was coupled with azido benzoic acid (5) and was

finally attached to the photocage using click chemistry to

generate the final Dox-EDANS conjugate (6).

The photolytic release of doxorubicin from the drug conjugate

was analyzed using HPLC. The drug conjugate was dissolved in

PBS buffer and was exposed to UV light at 365 nm (9.0 mW/cm2).

Aliquots of the reaction mixture were collected at various times

and were analyzed on RP-HPLC. During the course of the

reaction, the peaks corresponding to the Dox-EDANS conjugate

disappeared with concomitant increase in the intensity of a peak

with the same retention time as Dox (See ESI Fig. S3w), confirm-

ing time-depended drug release in the presence of light.

Fig. 1 Photocaged permeability strategy for drug delivery.

Fig. 2 (a) The anticancer drug doxorubicin. (b) Cell impermeable

EDANS.aDepartment of Chemistry, Virginia Commonwealth University(VCU), 1001 West Main Street, P. O. Box 842006, Richmond,VA 23284, USA. E-mail: mchartman@vcu.edu

bMassey Cancer Center, Virginia Commonwealth University,401 College Street, Richmond, VA 23298, USA

cDepartment of Pharmacology and Toxicology, VCU, Richmond,VA 23298, USA

dDepartment of Microbiology and Immunology, VCU, Richmond,VA 23298, USA

w Electronic supplementary information (ESI) available: Details ofsynthesis and characterization of the photocaged drug conjugate,photolytic drug release studies, cytotoxicity studies, confocal and flowcytometry data. See DOI: 10.1039/c2cc30819c

ChemComm Dynamic Article Links

www.rsc.org/chemcomm COMMUNICATION

Publ

ishe

d on

08

Mar

ch 2

012.

Dow

nloa

ded

by V

irgi

nia

Com

mon

wea

lth U

nive

rsity

on

02/1

0/20

13 1

4:06

:34.

View Article Online / Journal Homepage / Table of Contents for this issue

4756 Chem. Commun., 2012, 48, 4755–4757 This journal is c The Royal Society of Chemistry 2012

We then investigated whether the attachment of EDANS

to Dox via the veratryl linker would enable drug delivery.

JH-EsoAd1 cells, a Barrett’s esophagus associated adenocarci-

noma cell line,25 were incubated with Dox-EDANS in the dark

or with illumination. Cell permeability was measured with flow

cytometry; upon illumination a significant enhancement of

cellular Dox fluorescence was observed (Fig. 3A–B). This enhance-

ment was mirrored in confocal studies with the same cell line

(Fig. 3C)—only light-treated cells show significant Dox fluores-

cence in the nucleus, where it is known to accumulate.26

With permeability enhancement established, we proceeded

to investigate the extent to which the release of Dox with light

would lead to enhanced cellular toxicity. Indeed, increased

illumination lead to decreased survival as measured by an

MTT assay (Fig. 4). Survival of cells treated with EDANS-Dox

in the dark was equivalent to controls with no drug added.

Moreover, treatment with light alone at the same dose was not

cytotoxic (See ESI, Fig. S5w).

Finally, we sought to compare the concentration dependence

for our method vs. Dox alone. We first measured the IC50

of Dox alone with the JH-EsoAd1 cells and found it to be

1.0 � 0.4 mM (see ESI Fig. S6w). As expected by PCP,

EDANS-Dox was not toxic to the cells in the dark at the

highest value tested (16 mM). However, illumination of

Scheme 1 Synthesis of photocaged-cell impermeable drug conjugate.

Reagents and conditions: (a) NHS, EDC-HCl; (b) propargylamine, Et3N;

(c) bis p-nitrophenylcarbonate, Et3N; (d) doxorubicin-HCl, Et3N; (e)

NHS, EDC-HCl; (f) EDANS, EtN(iPr)2; (g) CuSO4�5H2O, sodium

ascorbate, tris-(benzyltriazolylmethyl)amine, DMSO:water (1 : 1).

Fig. 3 (A). The flow cytometry histogram displays the relative fluorescence intensity of untreated controls in the dark (blue) or light (red) or

JH-EsoAd1 cells treated with EDANS-Dox in the dark (orange) or light (green). (B) Quantification of Dox fluorescence for the indicated treatment

conditions. Data are representative of two independent experiments, N = 9. (C) Representative confocal images of JHEsoAd1 cells treated with

EDANS-Dox in the dark or light. The red channel shows Dox fluorescence, the gray channel is a DIC image, and the overlay represents the cellular

localization of Dox fluorescence.

Fig. 4 Light-dependent cytotoxicity of Dox-EDANS. Cells were treated

with 10 mM of Dox-EDANS with (solid diamonds) or without (open

squares) light for the specified times. After 2 h, the cells were washed

with fresh media and allowed to grow for 72 h. The fraction of surviving

cells was evaluated using the absorbance of the formazan product of

MTT reduction. Error bars denote one standard deviation from the

mean.

Publ

ishe

d on

08

Mar

ch 2

012.

Dow

nloa

ded

by V

irgi

nia

Com

mon

wea

lth U

nive

rsity

on

02/1

0/20

13 1

4:06

:34.

View Article Online

This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 4755–4757 4757

EDANS-Dox (365 nm, 9.0 mW/cm2) leads to cytotoxicity with

an IC50 of 1.6 � 1.0 mM (Fig. 5), comparable to that of Dox

alone. Based on our FACS study (Fig. 3), we deduce that this

enhanced cytotoxicity is caused by efficient light-stimulated

release of free Dox from the impermeable EDANS.

In conclusion, we have developed a new and efficient

strategy for drug release based on photocaged permeability

(PCP). In this first report, we have focused on applying PCP to

the light-stimulated delivery of Dox into esophageal adeno-

carcinoma cells. In principle, the PCP approach could be

applied to any small, cell permeable molecule that has a free

amine, hydroxyl, or carboxylic acid group for attachment of

the veratryl-EDANS molecule. Further experiments will focus

on use of other light-scissile linkers that can operate at longer

wavelengths that are able to penetrate farther into tissues, as

well as the use of other molecules that block permeability.

The authors acknowledge financial support from the

American Cancer Society (IRG-73-001-34) and the Massey

Cancer Center. Flow cytometry and confocal microscopy were

supported in part by grants P30CA16059 and 5P30NS047463.

We thank Dr M. Bertino (VCU) and M. Foussekis (VCU)

for help in measuring light intensity, Dr J. Eshleman (Johns

Hopkins) for the JH-EsoAd1 cells, and Dr A. Cropp (VCU)

for helpful comments.

Notes and references

1 F. Kratz, I. A. Muller, C. Ryppa and A. Warnecke, ChemMedChem,2008, 3, 20–53.

2 F. Fischel-Ghodsian, L. Brown, E. Mathiowitz, D. Brandenburgand R. Langer, Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 2403–2406.

3 G. M. Dubowchik, R. A. Firestone, L. Padilla, D. Willner, S. J.Hofstead, K. Mosure, J. O. Knipe, S. J. Lasch and P. A. Trail,Bioconjugate Chem., 2002, 13, 855–869.

4 M. Langer, F. Kratz, B. Rothen-Rutishauser, H. Wunderli-Allenspachand A. G. Beck-Sickinger, J. Med. Chem., 2001, 44, 1341–1348.

5 S. W. Choi, Y. Zhang and Y. Xia, Angew. Chem., Int. Ed., 2010,49, 7904–7908.

6 C. P. McCoy, C. Rooney, C. R. Edwards, D. S. Jones andS. P. Gorman, J. Am. Chem. Soc., 2007, 129, 9572–9573.

7 S. S. Agasti, A. Chompoosor, C. C. You, P. Ghosh, C. K. Kim andV. M. Rotello, J. Am. Chem. Soc., 2009, 131, 5728–5729.

8 S. K. Choi, T. Thomas, M. H. Li, A. Kotlyar, A. Desai andJ. R. Baker Jr, Chem. Commun., 2010, 46, 2632–2634.

9 O. V. Gerasimov, J. A. Boomer, M. M. Qualls andD. H. Thompson, Adv. Drug Delivery Rev., 1999, 38, 317–338.

10 H. M. Lee, D. R. Larson and D. S. Lawrence, ACS Chem. Biol.,2009, 4, 409–427.

11 A. Deiters, D. Groff, Y. Ryu, J. Xie and P. G. Schultz, Angew.Chem., Int. Ed., 2006, 45, 2728–2731.

12 A. Deiters, ChemBioChem, 2009, 11, 47–53.13 G. C. Ellis-Davies, Nat. Methods, 2007, 4, 619–628.14 B. N. Goguen, B. D. Hoffman, J. R. Sellers, M. A. Schwartz and

B. Imperiali, Angew. Chem., Int. Ed., 2011, 50, 5667–5670.15 C. W. Riggsbee and A. Deiters, Trends Biotechnol., 2010, 28,

468–475.16 P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti,

S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene,D. Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette,B. C. Wilson and J. Golab, Ca-Cancer J. Clin., 2011, 61, 250–281.

17 D. E. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer,2003, 3, 380–387.

18 K. Berg, A. Weyergang, L. Prasmickaite, A. Bonsted, A. Hogset,M. T. Strand, E. Wagner and P. K. Selbo, Methods Mol. Biol.,2010, 635, 133–145.

19 K. Berg, M. Folini, L. Prasmickaite, P. K. Selbo, A. Bonsted,B. O. Engesaeter, N. Zaffaroni, A. Weyergang, A. Dietze,G. M. Maelandsmo, E. Wagner, O. J. Norum and A. Hogset,Curr. Pharm. Biotechnol., 2007, 8, 362–372.

20 M. Verhille, P. Couleaud, R. Vanderesse, D. Brault, M. Barberi-Heyoband C. Frochot, Curr. Med. Chem., 2010, 17, 3925–3943.

21 Note: Drug delivery via light-stimulated release of cytotoxins fromcell impermeable polymers or nanoparticles has recently beendescribed (e.g. see ref. 6–8). Our PCP approach is distinct becausepermeability is blocked by a cell impermeable small molecule.

22 T. Fichert, M. Yazdanian and J. R. Proudfoot, Bioorg. Med.Chem. Lett., 2003, 13, 719–722.

23 C. P. Holmes, J. Org. Chem., 1997, 62, 2370–2380.24 G. Marriott, Biochemistry, 1994, 33, 9092–9097; R. Iwase,

A. Kitani, T. Yamaoka and A. Murakami, Nucleic Acids Symp.Ser., 2003, 3, 61–62; L. Baumann and A. G. Beck-Sickinger,Biopolymers, 2010, 94, 771–778; L. Berrade, Y. Kwon andJ. A. Camarero, ChemBioChem, 2010, 11, 1368–1372; R. Yang,K. K. Pasunooti, F. Li, X. W. Liu and C. F. Liu, Chem. Commun.,2010, 46, 7199–7201; J. P. Pellois, M. E. Hahn and T. W. Muir,J. Am. Chem. Soc., 2004, 126, 7170–7171; M. I. Sanchez,O. Vazquez, M. E. Vazquez and J. L. Mascarenas, Chem. Commun.,2011, 47, 11107–11109; S. Cho, S. H. Lee, W. J. Chung, Y. K. Kim,Y. S. Lee and B. G. Kim, Electrophoresis, 2004, 25, 3730–3739;Z. Gu, A. Biswas, K. I. Joo, B. Hu, P. Wang and Y. Tang, Chem.Commun., 2010, 46, 6467–6469; Y. Luo, N. V. Eldho, H. O. Sintimand T. K. Dayie, Nucleic Acids Res., 2011, 39, 8559–8571;J. B. Biggins, A. Hashimoto and J. T. Koh, ChemBioChem, 2007,8, 799–803; G. Mayer and A. Heckel, Angew. Chem., Int. Ed., 2006,45, 4900–4921.

25 A. Hector, J. M. Koorstra, S. Hong, J. J. Boonstra, W. N. Dinjens,A. A. Foratiere, T. Wu, E. A. Mongomery, J. R. Eshleman andA. Maitra, Cancer Biol. Ther., 2008, 7, 1753–1755.

26 G. Speelmans, R. W. Staffhorst, H. G. Steenbergen and B. deKruijff, Biochim. Biophys. Acta, Biomembr., 1996, 1284, 240–246.

Fig. 5 Concentration-dependent, light-stimulated cytotoxicity. Cells

were treated with EDANS-Dox at various concentrations in the dark

(open squares) or with UV light for 20 min (black diamonds). After 2 h,

the cells were washed with fresh media and allowed to grow for 72 h.

The fraction of surviving cells relative to no drug controls was

evaluated using the absorbance of the formazan product of MTT

reduction. Error bars denote one standard deviation from the mean.

Publ

ishe

d on

08

Mar

ch 2

012.

Dow

nloa

ded

by V

irgi

nia

Com

mon

wea

lth U

nive

rsity

on

02/1

0/20

13 1

4:06

:34.

View Article Online